Doxorubicin adjuvants to reduce toxicity and methods for using the same

ABSTRACT

Methods are provided for using doxorubicin active agents in which reduced host toxicity is observed. Aspects of the methods including administering to a subject an effective amount of a doxorubicin active agent in conjunction with a doxorubicin toxicity-reducing adjuvant, e.g., a nitrone compound, or a nitrone compound in combination with a bisdioxopiperazine compound. Also provided are compositions for use in practicing the subject methods. The methods and compositions find use in a variety of different applications, including in the treatment of a variety of different disease conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e), this application claims priority to thefiling date of the U.S. Provisional Patent Application Ser. No.61/028,090 filed Feb. 12, 2009; the disclosure of which is hereinincorporated by reference.

INTRODUCTION

Doxorubicin or hydroxyldaunorubicin is an antineoplastic drug widelyused in chemotherapy (Hortobágyi, Drugs, 54 (Supplement 4):1-7 (1997)).It is an anthracycline antibiotic and structurally closely related todaunomycin (Minotti et al., Pharmacologly. Rev., 56; 185-229 (2004).Doxorubicin (DOX) is commonly used in the treatment of a wide range ofcancers, including cancers of the blood, lymph system, bladder, breast,stomach, lung, ovaries, thyroid, nerves, kidneys, bones, soft tissues,including muscles and tendons, multiple myeloma, and others.

Doxorubicin is a highly toxic drug. Cardiotoxicity is its mostimportant, dose limiting toxicity (Outomuro et al., Int J Cardiol., 117:6-15 (2007)). As the cumulative dose of doxorubicin increases, the riskof developing cardiac side effects, including congestive heart failure,dilated cardiomyopathy and death, increases as well.

Attempts to minimize the toxicity of doxorubicin have includedcombination chemotherapy, synthesis of doxorubicin analogues, antibodyconjugates, immunotherapy and entrapment in Liposomes. One combinationemploys dexrazoxane (4-[1-(3,5-dioxopiperazin-1-yl)propan-2-yl]piperazine-2,6-dione), which is a cardioprotectant agentused to reduce the risk of cardiotoxicity (Hellmann, Semin Oncol, 25:48-54 (1998) and Hasinoff and Herman Cardioiovasc Toxicol, 7:140-144(2007). Liposomal formulations combining daunorubicin and doxorubicinalso appear to yield reduced cardiotoxicity (Batist, Cardiovasc Toxicol,7: 72-4 (2007). However, the problem has not been solved, and there iscontinued interest in finding new ways to reduce doxorubicin toxicity.

Nitrones are the N-oxide of imines first used as agents to trapfree-radicals (known as spin trapping) in chemical systems and,subsequently, in biochemical systems. Nitrones have been found to havepotential in the treatment of neurodegenerative diseases and otheraging-related diseases, such as stroke, Alzheimer's disease and thedevelopment of cancer. (Floyd et al., Free Radical Biology and Medicine,45, 1361-1374 (2008)). Besides decreasing oxidative stress and limitingoxidative damage, nitrones have also demonstrate anti-inflammatoryactivity in animal models of inflammation-associated diseases byaltering cellular signaling processes (Floyd et al., Free RadicalBiology and Medicine, 45, 1361-1374 (2008)).

α-phenyl-N-tert-butyl nitrone (“PBN”) is a widely researched nitronewhich has been found to have potent pharmacologic activities in variousneurodegenerative and aging-related disease models (Maples et al., CNSDrugs, 18(15), 1071-1084 (2004); Green et al., Pharmacol. Ther., 100(3),195-214 (2003); Floyd et al., Ann. N.Y. Acad. Sci., 959, 321-329 (2002);Floyd et al., Mech. Ageing Dev., 123(8), 1021-1031 (2002); Kotake,Antioxid. Redox Signal., 1(4), 481-499 (1999)).

SUMMARY

Methods of using doxorubicin active agents in which reduced hosttoxicity is observed are provided. In the subject methods, an effectiveamount of a doxorubicin active agent is administered to the host inconjunction with the administration of a doxorubicin toxicity-reducingadjuvant of the present invention, where the doxorubicin active agentand doxorubicin toxicity-reducing adjuvant may be administeredsequentially, simultaneously, or any combination thereof. Thedoxorubicin toxicity-reducing adjuvant is a compound containing anitrone functionality. Also provided are compositions for use inpracticing the subject methods, e.g., doxorubicin pharmaceuticalcompositions having reduced toxicity and kits that include the same.Compositions comprising thiol-modified nitrones also are provided thatfind use in the subject methods as well as other applications typicalof, or which benefit by the use of, nitrone compounds in general. Thus,the subject methods and compositions find use in a variety of differentapplications, including the treatment of a variety of different diseaseconditions. An exemplary application illustrating a significantadvantage of the methods and compositions of the invention is thereduction of doxorubicin-induced cardiac damage.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a set of data demonstrating the ability of TK-115339, arepresentative nitrone test article, to mitigate one aspect ofdoxorubicin-induced toxicity, cardiac damage in CD-1 mice as determinedby concentration of cardiac troponin I (cTnI) in animal plasma.

FIG. 2 depicts a set of data demonstrating a dose-response for theprotection afforded by TK-115339 against doxorubicin-induced toxicity.

FIG. 3 depicts a set of data demonstrating that TK-115339 does notinterfere with the toxicity of doxorubicin in CCRF-CEM human cancercells.

DEFINITIONS

When describing the compounds, pharmaceutical compositions containingsuch compounds and methods of using such compounds and compositions, thefollowing terms have the following meanings unless otherwise indicated.It should also be understood that any of the moieties defined forthbelow may be substituted with a variety of substituents, and that therespective definitions are intended to include such substituted moietieswithin their scope. By way of non-limiting example, such substituentsmay include e.g. halo (such as fluoro, chloro, bromo), —CN, —CF₃, —OH,—OCF₃, C₂₋₆ alkenyl, C₃₋₆ alkynyl, C₁₋₆ alkoxy, aryl and di-C₁₋₆alkylamino.

“Acyl” refers to a radical —C(O)R, where R is hydrogen, alkyl,cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroarylor heteroarylalkyl as defined herein. Representative examples include,but are not limited to, formyl, acetyl, cylcohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.

“Acylamino” refers to a radical —NR′C(O)R, where R′ is hydrogen, alkyl,cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl,heteroarylalkyl and R is hydrogen, alkyl, alkoxy, cycloalkyl,cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl orheteroarylalkyl, as defined herein. Representative examples include, butare not limited to, formylamino, acetylamino, cyclohexylcarbonylamino,cyclohexylmethyl-carbonylamino, benzoylamino, benzylcarbonylamino andthe like.

“Acyloxy” refers to the group —OC(O)H, —OC(O)-alkyl, —OC(O)-aryl or—OC(O)-cycloalkyl.

“Aliphatic” refers to hydrocarbyl organic compounds or groupscharacterized by a straight, branched or cyclic arrangement of theconstituent carbon atoms and an absence of aromatic unsaturation.Aliphatics include, without limitation, alkyl, alkylene, alkenyl,alkenylene, alkynyl and alkynylene. Aliphatic groups typically have from1 or 2 to 6 or 12 carbon atoms.

“Alkanoyl” or “acyl” as used herein refers to the group —C(O)H or—C(O)-alkyl.

“Alkenyl” refers to monovalent olefinically unsaturated hydrocarbylgroups having up to about 11 carbon atoms, particularly, from 2 to 8carbon atoms, and more particularly, from 2 to 6 carbon atoms, which canbe straight-chained or branched and having at least 1 and particularlyfrom 1 to 2 sites of olefinic unsaturation. Particular alkenyl groupsinclude ethenyl (—CH═CH₂), n-propenyl (—CH₂CH═CH₂), isopropenyl(—C(CH₃)═CH₂), vinyl and substituted vinyl, and the like.

“Alkenylene” refers to divalent olefinically unsaturated hydrocarbylgroups particularly having up to about 11 carbon atoms and moreparticularly 2 to 6 carbon atoms which can be straight-chained orbranched and having at least 1 and particularly from 1 to 2 sites ofolefinic unsaturation. This term is exemplified by groups such asethenylene (—CH═CH—), the propenylene isomers (e.g., —CH═CHCH₂— and—C(CH₃)═CH— and —CH═C(CH₃)—) and the like.

“Alkoxy” refers to the group —O-alkyl. Particular alkoxy groups include,by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, andthe like.

“Alkoxyamino” refers to a radical —N(H)O-alkyl or —N(H)O-cycloalkyl asdefined herein.

“Alkoxycarbonyl” refers to a radical —C(O)-alkoxy where alkoxy is asdefined herein.

“Alkoxycarbonylamino” refers to the group —NRC(O)OR′ where R ishydrogen, alkyl, aryl or cycloalkyl, and R′ is alkyl or cycloalkyl.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupsparticularly having up to about 11 carbon atoms, more particularly as alower alkyl, from 1 to 8 carbon atoms and still more particularly, from1 to 6 carbon atoms. The hydrocarbon chain may be eitherstraight-chained or branched. This term is exemplified by groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl,n-hexyl, n-octyl, tert-octyl and the like. The term “lower alkyl” refersto alkyl groups having 1 to 6 carbon atoms. The term “alkyl” alsoincludes “cycloalkyls” as defined below.

“Alkylamino” refers to a radical alkyl-NRR′, wherein each of R and R′are independently selected from hydrogen and alkyl.

“Alkylarylamino” refers to a radical —NRR′ where R represents an alkylor cycloalkyl group and R′ is an aryl as defined herein.

“Alkylene” refers to divalent saturated aliphatic hydrocarbyl groupsparticularly having up to about 11 carbon atoms and more particularly 1to 6 carbon atoms which can be straight-chained or branched. This termis exemplified by groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),the propylene isomers (e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

“Alkylthio” refers to a radical —S-alkyl or —S-cycloalkyl group asdefined herein that may be optionally substituted as defined herein.Representative examples include, but are not limited to, methylthio,ethylthio, propylthio, butylthio, and the like.

“Alkynyl” refers to acetylenically unsaturated hydrocarbyl groupsparticularly having up to about 11 carbon atoms and more particularly 2to 6 carbon atoms which can be straight-chained or branched and havingat least 1 and particularly from 1 to 2 sites of alkynyl unsaturation.Particular non-limiting examples of alkynyl groups include acetylenic,ethynyl (—C≡CH), propargyl (—CH₂C≡CH), and the like.

“Amino” refers to the radical —NH₂.

“Aminocarbonyl” refers to the group —C(O)NRR where each R isindependently hydrogen, alkyl, aryl or cycloalkyl, or where the R groupsare joined to form an alkylene group.

“Aminocarbonylamino” refers to the group —NRC(O)NRR where each R isindependently hydrogen, alkyl, aryl or cycloalkyl, or where two R groupsare joined to form an alkylene group.

“Aminocarbonyloxy” refers to the group —OC(O)NRR where each R isindependently hydrogen, alkyl, aryl or cycloalky, or where the R groupsare joined to form an alkylene group.

“Aminohydroxyphosphoryl” refers to the radical —PO(OH)NH₂.

“Aralkyl” or “arylalkyl” refers to an alkyl group, as defined above,substituted with one or more aryl groups, as defined above.

“Aryl” refers to a monovalent aromatic hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene and the like. Particularly, anaryl group comprises from 6 to 14 carbon atoms.

“Arylalkyloxy” refers to an —O-arylalkyl radical where arylalkyl is asdefined herein.

“Arylamino” refers to the group aryl-NRR′, wherein each of R and R′ areindependently selected from hydrogen, aryl and heteroaryl.

“Aryloxy” refers to —O-aryl groups wherein “aryl” is as defined herein.

“Arylsulfonyl” refers to a radical —S(O)₂R where R is an aryl orheteroaryl group as defined herein.

“Azido” refers to the radical —N₃.

“Carbamoyl” refers to the radical —C(O)N(R)₂ where each R group isindependently hydrogen, alkyl, cycloalkyl or aryl, as defined herein,which may be optionally substituted as defined herein.

“Carboxy” refers to the radical —C(O)OH.

“Cyano” refers to the radical —CN.

“Cycloalkenyl” refers to cyclic hydrocarbyl groups having from 3 to 10carbon atoms and having a single cyclic ring or multiple condensedrings, including fused and bridged ring systems and having at least oneand particularly from 1 to 2 sites of olefinic unsaturation. Suchcycloalkenyl groups include, by way of example, single ring structuressuch as cyclohexenyl, cyclopentenyl, cyclopropenyl, and the like.

“Cycloalkyl” refers to cyclic hydrocarbyl groups having from 3 to about10 carbon atoms and having a single cyclic ring or multiple condensedrings, including fused and bridged ring systems, which optionally can besubstituted with from 1 to 3 alkyl groups. Such cycloalkyl groupsinclude, by way of example, single ring structures such as cyclopropyl,cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl,2-methylcyclopentyl, 2-methylcyclooctyl, and the like, and multiple ringstructures such as adamantanyl, and the like.

“Cycloheteroalkyl” refers to a stable heterocyclic non-aromatic ring andfused rings containing one or more heteroatoms independently selectedfrom N, O and S. A fused heterocyclic ring system may includecarbocyclic rings and need only include one heterocyclic ring. Examplesof heterocyclic rings include, but are not limited to, piperazinyl,homopiperazinyl, piperidinyl and morpholinyl, and are shown in thefollowing illustrative examples:

optionally substituted with one or more groups selected from the groupconsisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy,alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen,hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂- and aryl-S(O)₂—.Substituting groups include carbonyl or thiocarbonyl which provide, forexample, lactam and urea derivatives. In the examples, M is CR⁷, NR³, O,or S; Q is O, NR³ or S.

“Dialkylamino” means a radical —NRR′ where R and R′ independentlyrepresent an alkyl, substituted alkyl, aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substitutedcycloheteroalkyl, heteroaryl, or substituted heteroaryl group as definedherein.

“Dihydroxyphosphoryl” refers to the radical —PO(OH)₂.

“Doxorubicin pharmaceutical composition” refers to a doxorubicin activeagent used in combination with a doxorubicin toxicity-reducing adjuvant,either to be administered separately on in a combined formulation.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo. Halogroups can be either fluoro or chloro.

“Hetero” when used to describe a compound or a group present on acompound means that one or more carbon atoms in the compound or grouphave been replaced by a nitrogen, oxygen, or sulfur heteroatom. Heteromay be applied to any of the hydrocarbyl groups described above such asalkyl, e.g. heteroalkyl, cycloalkyl, e.g. cycloheteroalkyl, aryl, e.g.heteroaryl, cycloalkenyl, cycloheteroalkenyl, and the like having from 1to 5, and especially from 1 to 3 heteroatoms. Examples of representativecycloheteroalkenyls include the following:

wherein each X is selected from CR⁵, NR⁵, O and S; and each Y isselected from carbonyl, N, NR⁵, O and S.

“Heteroaryl” refers to a monovalent heteroaromatic group derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. In some embodiments, theheteroaryl group is a 5-20 membered heteroaryl, or 5-10 memberedheteroaryl. Particular heteroaryl groups are those derived fromthiophen, pyrrole, benzothiophene, benzofuran, indole, pyridine,quinoline, imidazole, oxazole and pyrazine. Examples of representativeheteroaryls include the following:

wherein each Y is selected from carbonyl, N, NR⁵, O, and S. Examples ofrepresentative aryl having hetero atoms containing substitution includethe following:

wherein each X is selected from C—R⁵, C(R⁵)₂, NR⁵, O and S; and each Yis selected from carbonyl, NR⁵, O and S.

“Hydroxyl” refers to the radical —OH.

“Nitro” refers to the radical —NO₂.

“R¹” is each independently selected from the group consisting ofsubstituted or unsubstituted aliphatic, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted aralkyl, and substituted or unsubstitutedheteroaralkyl.

“R²” and “R³” are each independently selected from the group consistingof hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, and substituted or unsubstituted aralkyl.

“R⁴” is each independently selected from the group consisting ofhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted aralkyl, and any two R⁴s may join togetherto form a cycloalkyl, cycloheteroalkyl ring.

“R⁵” is each independently hydrogen, alkyl, substituted alkyl, acyl,substituted acyl, acylamino, substituted acylamino, alkylamino,substituted alkylamino, alkylthio, substituted alkylthio, alkoxy,substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl,alkylarylamino, substituted alkylarylamino, arylalkyloxy, substitutedarylalkyloxy, amino, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, sulfoxide, substituted sulfoxide, sulfone, substitutedsulfone, sulfanyl, substituted sulfanyl, aminosulfonyl, substitutedaminosulfonyl, arylsulfonyl, substituted arylsulfonyl, sulfonic acid,sulfonic acid ester (i.e., sulfonate), dihydroxyphosphoryl, substituteddihydroxyphosphoryl, aminohydroxyphosphoryl, substitutedaminohydroxyphosphoryl, azido, carboxy, substituted carboxy (i.e.,ester), carbamoyl, substituted carbamoyl, cyano, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl,dialkylamino, substituted dialkylamino, halo, heteroaryloxy, substitutedheteroaryloxy, heteroaryl, substituted heteroaryl, heteroalkyl,substituted heteroalkyl, hydroxyl, nitro or thio.

“R⁶” is selected from the group consisting of hydrogen, —SR⁷, —SO₂R⁷,SO₂NR⁷R⁸, —SO₃R⁷, —CONR⁷R⁸, —NR⁷R⁸, —OH, —PO(OR⁷)NR⁸R⁹, —PO(OR⁷)₂ and—CO₂R⁷.

“R⁷”, “R⁸”, and “R⁹” are independently selected from the groupconsisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy,alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen,hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂- and aryl-S(O)₂—.

“R¹⁰”, “R¹¹”, and “R¹²” are independently hydrogen, alkyl, alkenyl,alkynyl, perfluoroalkyl, cycloalkyl, cycloheteroalkyl, aryl, substitutedaryl, heteroaryl, substituted or hetero alkyl or the like.

“R¹³” and “R²⁰” are independently hydrogen, alkyl, alkenyl, alkynyl,cycloheteroalkyl, alkanoyl, alkoxy, aryloxy, heteroaryloxy, alkylamino,arylamino, heteroarylamino, NR¹⁰COR¹¹, NR¹⁰SOR¹¹, NR¹⁰SO₂R¹⁴, COOalkyl,COOaryl, CONR¹⁰R¹¹, CONR¹⁰OR¹¹, NR¹⁰R¹¹, SO₂NR¹⁰R¹¹, S-alkyl, S-alkyl,SOalkyl, SO₂alkyl, Saryl, SOaryl, or SO₂aryl; or R¹³ and R²⁰ form acyclic ring (saturated or unsaturated) from 5 to 8 atoms, optionallycontaining one or more heteroatoms selected from the group N, O or S.

“R¹⁴”, “R¹⁵”, “R¹⁶”, and “R¹⁷” are independently hydrogen, alkyl,substituted alkyl, aryl, arylalkyl, cycloalkyl, cycloheteroalkyl,substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl,heteroaryl, substituted heteroaryl, heteroarylalkyl, substitutedheteroarylalkyl, —NR¹⁸R¹⁹, —C(O)R¹⁸ or —S(O)₂R¹⁸ or optionally R¹⁸ andR¹⁹ together with the atom to which they are both attached form acycloheteroalkyl or substituted cycloheteroalkyl ring.

“R¹⁸”, “R¹⁹” and “R²²” are each independently selected from the groupconsisting of hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted aryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedcycloheteroalkyl, substituted or unsubstituted heteroalkyl, substitutedor unsubstituted heteroaryl, and substituted or unsubstitutedheteroarylalkyl.

“R²¹” is R²² or —S—R²².

“Spatial isomers” refers to isomers other than structural isomers.Examples include, but are not limited to, stereoisomers such asenantiomers, diastereomers, geometric isomers, tautomers, cis-transisomers, isotopic isomers, and spin isomers.

“Substituted” refers to a group in which one or more hydrogen atoms areeach independently replaced with the same or different substituent(s).Typical substituents include, but are not limited to, —X, —R¹⁴, —O—, ═O,—OR¹⁴, —SR¹⁴, —S⁻, ═S, —NR¹⁴R¹⁵, ═NR¹⁴, —CX₃, —CF₃, —CN, —OCN, —SCN,—NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R¹⁴, —OS(O₂)O⁻,—OS(O)₂R¹⁴, —P(O)(O—)₂, —P(O)(OR¹⁴)(O), —OP(O)(OR¹⁴)(OR¹⁵), —C(O)R¹⁴,—C(S)R¹⁴, —C(O)OR¹⁴, —C(O)NR¹⁴R¹⁵, —C(O)O⁻, —C(S)OR¹⁴, —NR¹⁶C(O)NR¹⁴R¹⁵,—NR¹⁶C(S)NR¹⁴R¹⁵, —NR¹⁷C(NR¹⁶)NR¹⁴R¹⁵ and —C(NR¹⁶)NR¹⁴R¹⁵, where each Xis independently a halogen.

“Substituted alkenyl” includes those groups recited in the definition of“substituted” herein, and particularly refers to an alkenyl group having1 or more substituents, for instance from 1 to 5 substituents, andparticularly from 1 to 3 substituents, selected from the groupconsisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy,alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen,hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— and aryl-S(O)₂—.

“Substituted alkoxy” includes those groups recited in the definition of“substituted” herein, and particularly refers to an alkoxy group having1 or more substituents, for instance from 1 to 5 substituents, andparticularly from 1 to 3 substituents, selected from the groupconsisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy,alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen,heteroaryl, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy,thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— andaryl-S(O)₂—.

“Substituted alkyl” includes those groups recited in the definition of“substituted” herein, and particularly refers to an alkyl group having 1or more substituents, for instance from 1 to 5 substituents, andparticularly from 1 to 3 substituents, selected from the groupconsisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy,alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen,hydroxyl, heteroaryl, keto, nitro, thioalkoxy, substituted thioalkoxy,thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂—, andaryl-S(O)₂—.

“Substituted alkylene” includes those groups recited in the definitionof “substituted” herein, and particularly refers to an alkylene grouphaving 1 or more substituents, for instance from 1 to 5 substituents,and particularly from 1 to 3 substituents, selected from the groupconsisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy,alkoxycarbonyl, alkoxycarbonylannino, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, halogen, hydroxyl, keto, nitro, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)—,aryl-S(O)—, alkyl-S(O)₂— and aryl-S(O)₂—.

“Substituted alkynyl” includes those groups recited in the definition of“substituted” herein, and particularly refers to an alkynyl group having1 or more substituents, for instance from 1 to 5 substituents, andparticularly from 1 to 3 substituents, selected from the groupconsisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy,alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen,hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— and aryl-S(O)₂—.

“Substituted amino” includes those groups recited in the definition of“substituted” herein, and particularly refers to the group —N(R)₂ whereeach R is independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, cycloalkyl, substituted cycloalkyl, and whereboth R groups are joined to form an alkylene group.

“Substituted aminohydroxyphosphoryl” includes those groups recited inthe definition of “substituted” herein, and particularly refers to anaminohydroxyphosphoryl wherein the amino group is substituted with oneor two substituents. In certain embodiments, the hydroxyl group can alsobe substituted.

“Substituted aryl” includes those groups recited in the definition of“substituted” herein, and particularly refers to an aryl group that mayoptionally be substituted with 1 or more substituents, for instance from1 to 5 substituents, particularly 1 to 3 substituents, selected from thegroup consisting of acyl, acylamino, acyloxy, alkenyl, substitutedalkenyl, alkoxy, substituted alkoxy, alkoxycarbonyl, alkyl, substitutedalkyl, alkynyl, substituted alkynyl, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen,hydroxyl, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thiol,alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— and aryl-S(O)₂—. Examples ofrepresentative substituted aryls include the following structures:

“Substituted cycloalkenyl” includes those groups recited in thedefinition of “substituted” herein, and particularly refers to acycloalkenyl group having 1 or more substituents, for instance from 1 to5 substituents, and particularly from 1 to 3 substituents, selected fromthe group consisting of acyl, acylamino, acyloxy, alkoxy, substitutedalkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen,hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— and aryl-S(O)₂—.

“Substituted cycloalkyl” includes those groups recited in the definitionof “substituted” herein, and particularly refers to a cycloalkyl grouphaving 1 or more substituents, for instance from 1 to 5 substituents,and particularly from 1 to 3 substituents, selected from the groupconsisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy,alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen,hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— and aryl-S(O)₂—.

“Substituted dihydroxyphosphoryl” includes those groups recited in thedefinition of “substituted” herein, and particularly refers to adihydroxyphosphoryl radical wherein one or both of the hydroxyl groupsare substituted.

“Substituted thioalkoxy” includes those groups recited in the definitionof “substituted” herein, and particularly refers to a thioalkoxy grouphaving 1 or more substituents, for instance from 1 to 5 substituents,and particularly from 1 to 3 substituents, selected from the groupconsisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy,alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen,hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— and aryl-S(O)₂.

“Sulfanyl” refers to the radical —SH. “Substituted sulfanyl” refers to aradical such as —SR wherein R is any substituent described herein.

“Sulfone” refers to the group —SO₂R. In particular embodiments, R isselected from H, lower alkyl, alkyl, aryl and heteroaryl.

“Sulfonyl” refers to the divalent radical —S(O₂)—. “Substitutedsulfonyl” refers to a radical such as R—(O₂)S— wherein R is anysubstituent described herein. “Aminosulfonyl” refers to the radicalH₂N(O₂)S—, and “substituted aminosulfonyl” refers to a radical such asR₂N(O₂)S— wherein each R is independently any substituent describedherein.

“Thioalkoxy” refers to the group —S-alkyl.

“Thioaryloxy” refers to the group —S-aryl.

“Thioketo” refers to the group ═S.

“Thiol” refers to the group —SH.

One having ordinary skill in the art will recognize that the maximumnumber of heteroatoms in a stable, chemically feasible heterocyclicring, whether it is aromatic or non aromatic, is determined by the sizeof the ring, the degree of unsaturation and the valence of theheteroatoms. In general, a heterocyclic ring may have one to fourheteroatoms so long as the heteroaromatic ring is chemically feasibleand stable.

DETAILED DESCRIPTION

Methods are provided for using doxorubicin active agents in whichreduced host toxicity is observed. In the subject methods, an effectiveamount of a doxorubicin active agent is administered to the host inconjunction with the administration of a doxorubicin toxicity-reducingadjuvant of the present invention, where the doxorubicin active agentand doxorubicin toxicity-reducing adjuvant may be administeredsequentially, simultaneously, or any combination thereof. Also providedare compositions for use in practicing the subject methods, e.g.,doxorubicin pharmaceutical compositions having reduced toxicity and kitsthat include the same. Compositions comprising thiol-modified nitronesalso are provided that find use in the subject methods as well as otherapplications typical of, or which benefit by the use of, nitronecompounds in general. The subject methods and compositions find use in avariety of different applications, including the treatment of a varietyof different disease conditions. An exemplary application illustrating asignificant advantage of the methods and compositions of the inventionis the reduction of doxorubicin-induced cardiac damage

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

In further describing the subject invention, the subject methods aredescribed first in greater detail, followed by a review of the variouscompositions, e.g., formulations and kits, that may find use in thesubject methods, as well as a discussion of various representativeapplications in which the subject methods and compositions find use.

Methods

As summarized above, the subject invention provides methods ofadministering a doxorubicin active agent to a subject in need thereof,e.g., for the treatment of a host suffering from disease or conditiontreatable by a doxorubicin active agent (as described in greater detailbelow). An aspect of the subject methods is that the doxorubicin activeagent is administered to the subject in combination with a doxorubicintoxicity-reducing adjuvant which is a nitrone compound. By “incombination with” is meant that an amount of the doxorubicintoxicity-reducing adjuvant is administered anywhere from simultaneouslyto up to 5 hours or more, e.g., 10 hours, 15 hours, 20 hours or more,prior to or after the doxorubicin active agent. In certain embodiments,the doxorubicin active agent and doxorubicin toxicity-reducing adjuvantare administered sequentially, e.g., where the doxorubicin active agentis administered before or after the doxorubicin toxicity-reducingadjuvant. In yet other embodiments, the doxorubicin active agent anddoxorubicin toxicity-reducing adjuvant are administered simultaneously,e.g., where the doxorubicin active agent and doxorubicintoxicity-reducing adjuvant are administered at the same time as twoseparate formulations or are combined into a single composition that isadministered to the subject. Regardless of whether the doxorubicinactive agent and doxorubicin toxicity-reducing adjuvant are administeredsequentially or simultaneously, as illustrated above, the agents areconsidered to be administered together or in combination for purposes ofthe present invention. Routes of administration of the two agents mayvary, where representative routes of administration are described ingreater detail below.

In the subject methods, an effective amount of a doxorubicin activeagent is administered to a host in need thereof in combination with aneffective amount of a doxorubicin toxicity-reducing adjuvant. Bydoxorubicin active agent is meant doxorubicin or an analogue/derivativethereof, e.g., native doxorubicin and its analogues. Doxorubicin is ananthracycline antibiotic first isolated from the fungus Streptomycespeucetius. The chemical structure of doxorubicin consists of atetracyclic ring, with the sugar daunosamine attached by a glycosidiclinkage. Structurally, doxorubicin is related to daunomycin(daunorubicin) and differs only in hydroxyl group substitution (insteadof hydrogen) at the alkyl side chain, at position ‘9’ of the ‘A’ ring.However, daunorubicin is only useful for acute leukemia whereasdoxorubicin can be used for a wide range of cancers. (See, “doxorubicin”content at www.fda.gov, in the 2007 Physicians Desk Reference and inother similar references). The hydrochloride salt of doxorubicin is oneof the most common forms. It is referred to by various names, such asdoxorubicin hydrochloride; 14-hydroxydaunorubicin hydrochloride;3-hydroxyacetyldaunorubicin hydrochloride; and 5,12-naphthacenedione,10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-,hydrochloride,(8S-cis)-(9Cl). Doxorubicin hydrochloride has the molecular formulaC₂₇H₂₉NO₁₁.HCl, a molecular weight (MW) of 580.0, and CAS number25316-40-9. It is soluble in water and slightly soluble in methanol.

Doxorubicin can be thought of as a prototype compound for theanthracyclines daunorubicin, epirubicin and idarubicin. Although manyderivatives of doxorubicin have been made in an attempt to reproduce thesame or improved anti-tumor effects with less cardiac toxicity,doxorubicin remains the most widely administered of the anthracyclines.Nevertheless, a wide spectrum of analogues have been synthesized,offering a different antitumor spectrum, better therapeutic index andreduced toxicity than that offered by native doxorubicin (See, e.g.,Weiss, R B, Semin Oncol., 19:670 (1992)). For example, analogues ofdoxorubicin have been made with similar to as much as 1000 times theanti-proliferative activity of the native compound, some withsignificantly reduced toxicity, and others with both attributes ofanti-proliferative activity and reduced toxicity (Nagy et al., Proc NatlAcad Sci USA. 93 (6):2464 (1996); Nagy et al., Proc Natl Acad Sci USA.95 (4):1794 (1998); Wasowska et al., Anticancer Res. 25(3B):2043 (2005);Fan et al., J Org Chem. 72(8):2917 (2007); Zhang et al., J Med Chem.49(5):1792 (2006); Battisti et al., Mol Pharm. 4(1):140 (2007); Fang etal., J Med Chem. 49(3):932 (2006); Partugal at al., J Med Chem.48(26):8209 (2005); Haj et al., Chem Biol Interact. 145(3):349 (2003);Suarato at al., Curr Pharm Des. 5(3):217 (1999); Chaires et al., J MedChem. 40(3):261 (1997); and Ripamonti et al., Invest New Drugs.14(2):139 (1996)). While more toxic analogues are not desirable forintravenous administration in free form, such analogues may have use inLiposome-entrapped forms, which reduces drug toxicity.

Doxorubicin active agents of the present invention include doxorubicinand any analogues or derivatives thereof whose toxicity is reduced whenadministered in conjunction with a toxicity-reducing adjuvant accordingto the subject invention. Whether or not a given doxorubicin activeagent is suitable for use according to the present invention can bereadily determined using assays employed in the experimental section,below. Generally, a doxorubicin active agent is suitable for use in thesubject methods if its toxicity is reduced by 2-fold or more, such as by10-fold or more and including by 100-fold or more, which can bedetermined in vitro and/or in vivo as described in the Experimentalsection, below. In certain embodiments, the doxorubicin active agent isone that reduces the occurrence and/or intensity of observable toxicside effects as observed in the mouse assay described in theExperimental section below.

By doxorubicin toxicity-reducing adjuvant it is meant an agent thatreduces unwanted toxicity of a doxorubicin active agent.Toxicity-reducing adjuvants of interest are those agents that reduce thetoxicity of a doxorubicin active agent by 2-fold or more, such as by10-fold and including by 100-fold or more, which can be determined invitro and/or in vivo as described in the Experimental section, below. Incertain embodiments, the toxicity-reducing adjuvants of interest arethose that reduce the occurrence and/or intensity of observable toxicside effects of a given doxorubicin active agent, as observed in themouse assay described in the Experimental section below.

The doxorubicin toxicity-reducing adjuvants of interest include nitronecompounds. In some embodiments, the nitrone is a compound of formula(I):

or a nitrone compound of formula (I) according to formula (II):

or a nitrone compound of formula (II) according to formula (III):

or the pharmaceutically acceptable salts, solvates, hydrates, andprodrug forms thereof, and spatial isomers thereof;

wherein:

L is —[C(R³)₂]_(m)—X*—[C(R⁴)₂]_(n)—; m is an integer from 0 to 6; n isan integer from 0 to 6;

X* is selected from the group consisting of no atom, NR³, O, S, SO andSO₂;

R is hydrogen, thiol, or a thiol conjugate;

each Cy is independently selected from the group consisting ofsubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted cycloheteroalkyl, bicycloalkenyl, bicycloheteroalkenyl,bicycloaryl, or bicycloheteroaryl ring;

each R¹ is independently selected from the group consisting ofsubstituted or unsubstituted aliphatic, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted aralkyl, and substituted or unsubstitutedheteroaralkyl;

each R² is independently selected from the group consisting of hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, and substituted or unsubstituted aralkyl;

each R³ is independently selected from the group consisting of hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, and substituted or unsubstituted aralkyl;

each R⁴ is independently selected from the group consisting of hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted aralkyl, and any two R⁴s may join together to form acycloalkyl, cycloheteroalkyl ring;

and one of R³s and one of R⁴s on carbon atoms adjacent to X* may jointogether to form a heterocyclic ring of 5-7 atoms.

While the nitrone compounds in formulas (I), (II) and (III) depict oneisomer of the carbon-nitrogen double bond of the nitronefunctionalities, the scope of the present invention includes allgeometric isomers of the nitrone compounds of formulas (I), (II) and(II) including, for example, all isomers (e.g., E and Z isomers) of thecarbon-nitrogen double bond of each nitrone functionality. Eachstructure shown encompasses or represents both, or any one of, the E andZ isomers, or a mixture thereof.

With respect to the above formulas, it is noted that structures (I),(II) and (III) may also be written where the nitrone moiety does notinclude a double bonde between the O and N, such that N carries apositive charge and O carries a negative charge. For example, structureII may be written as structure II(a):

Alternatively, the nitrone moiety be represented by a structure in whichan arrow points from the N to the O, as shown in certain structuresbelow.

In certain embodiments, the present invention provides aryl,heteroaromatic and bicyclic aryl nitrone compounds according to formulas(I), (II) or (III), and wherein Cy is

and wherein;

for aryl nitrones, W and Z are joined to form a substituted orunsubstituted cycloalkenyl or aryl ring of 5 to 8 atoms; forheteroaromatic nitrones, W and Z are joined to form a substituted orunsubstituted cycloheteroalkenyl or heteroaryl ring of 5 to 8 atoms; andfor bicyclic aryl nitrones, W and Z are joined to form a bicycloalkenyl,bicycloheteroalkenyl, bicycloaryl, or bicycloheteroaryl ring of 8 to 11atoms.

In certain embodiments, the present invention provides aryl andheteroaromatic nitrone compounds according to (I), (II) and (III) andwherein Cy is

wherein: m′ of W, W′, X, Y and Z is N and the remainder are eachindependently C—R⁵; and m′ is an integer from 0 to 3.

In certain embodiments, the present invention provides heteroaromaticnitrone compounds according to formulas (I), (II) or (III) and whereinCy is

wherein: W, W′, X, and Z is independently selected from C—R⁵, O, S, SO,SO₂, NR³ and N; and the dotted bond is single or double bond.

In certain embodiments, the present invention provides bicyclic arylnitrone compounds according to formulas (I), (II) or (III) and whereinCy is

wherein W, W′, X, Y and Z are members of a cycloalkenyl, aryl,cycloheteroalkenyl or heteroaryl ring; and any adjacent pair of W, W′,X, Y and Z are further joined to form, together with the cycloalkenyl,aryl, cycloheteroalkenyl or heteroaryl ring comprising W, W′, X, Y andZ, the bicycloalkenyl, bicycloheteroalkenyl, bicycloaryl, orbicycloheteroaryl ring.

In certain embodiments, the present invention provides bicyclic arylnitrone compounds according to formulas (I), (II) or (III) and whereinCy is selected from substituted or unsubstituted:

and wherein A, Y and Z are independently selected from C═O, CR⁵, NR³, O,and S; and the dotted line represents single or double bond.

In certain embodiments, the present invention provides bicyclic arylnitrone compounds according to formulas (I), (II) or (III) and whereinCy is selected from substituted or unsubstituted:

wherein W, W′, X and X′ are each independently NR³ or C—R⁵; Y and Z areeach independently C—R⁵ or carbonyl; A and Q are independently selectedfrom C—R⁵, NR³, O, and S; and the dotted line represents single ordouble bond.

In certain embodiments, the present invention provides bicyclic arylnitrone compounds according to formulas (I), (II) or (III) and whereinCy is selected from substituted or unsubstituted:

In certain embodiments, the present invention provides nitrone compoundsaccording to formulas (I), (II) or (III) and wherein R² is hydrogen.

In certain embodiments, the present invention provides nitrone compoundsaccording to formulas (I), (II) or (III) and wherein L is—[C(R³)₂]_(m)—X*—[C(R⁴)₂]_(n)—.

In further embodiments, the present invention provides nitrone compoundsaccording to formulas (I), (II) or (III) and wherein L is—[CH₂]_(m)—X*—[CH₂]_(n)—.

In further embodiments, the present invention provides nitrone compoundsaccording to formulas (I), (II) or (III) and wherein L is selected from—CH₂—, —(CH₂)₂—, —(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅—, —OCH₂—, —O(CH₂)₂—,—O(CH₂)₃—, —O(CH₂)₄—, —O(CH₂)₅—, —SCH₂—, —S(CH₂)₂—, —S(CH₂)₃—,—S(CH₂)₄—, —S(CH₂)₅—, —SOCH₂—, —SO(CH₂)₂—, —SO(CH₂)₃—, —SO(CH₂)₄—,—SO(CH₂)₅—, —N(Me)CH₂—, —SO₂CH₂—, —SO₂(CH₂)₂—, —SO₂(CH₂)₃—, —SO₂(CH₂)₄—,—SO₂(CH₂)₅—, —N(Me)(CH₂)₂—, —N(Me)(CH₂)₃—, —N(Me)(CH₂)₄—, —N(Me)(CH₂)₅—,—CH₂—O—CH₂—, —CH₂—O—(CH₂)₂—, —CH₂—O—(CH₂)₃—, —(CH₂)₂—O—CH₂—,—(CH₂)₂—O—(CH₂)₂—, —(CH₂)₃—O—CH₂—, —(CH₂)₃—O—(CH₂)₂—, —CH₂—S—CH₂—,—CH₂—S—(CH₂)₂—, —CH₂—S—(CH₂)₃—, —(CH₂)₂—S—CH₂—, —(CH₂)₂—S—(CH₂)₂—,—(CH₂)₃—S—CH₂—, —(CH₂)₃—S—(CH₂)₂—, —CH₂—SO—CH₂—, —CH₂—SO—(CH₂)₂—,—CH₂—SO—(CH₂)₃—, —(CH₂)₂—SO—CH₂—, —(CH₂)₂—SO—(CH₂)₂—, —(CH₂)₃—SO—CH₂—,—(CH₂)₃—SO—(CH₂)₂—, —CH₂—SO₂—CH₂—, —CH₂—SO₂—(CH₂)₂—, —CH₂—SO₂—(CH₂)₃—,—(CH₂)₂—SO₂—CH₂—, —(CH₂)₂—SO₂—(CH₂)₂—, —(CH₂)₃—SO₂—CH₂—,—(CH₂)₃—SO₂—(CH₂)₂—, —CH₂—N(Me)-CH₂—, —CH₂—N(Me)-(CH₂)₂—,—CH₂—N(Me)-(CH₂)₃—, —(CH₂)₂—N(Me)-CH₂—, —(CH₂)₂—N(Me)-(CH₂)₂—,—(CH₂)₃-N(Me)-CH₂—, and —(CH₂)₃—N(Me)-(CH₂)₂—.

In further embodiments, the present invention provides nitrone compoundsaccording to formulas (I), (II) or (III) and wherein L is no atom.

In certain embodiments, the present invention provides nitrone compoundsaccording to formulas (I), (II) or (III) and wherein R¹ is Pert-butyl.

In certain embodiments, the present invention provides nitrone compoundsaccording to formulas (I), (II) or (III) and wherein R¹ is cyclohexyl.

In certain embodiments, the present invention provides nitrone compoundsaccording to formulas (I), (II) or (III) and wherein R¹ is benzyl.

Among the aryl nitrones described above by formula (I), (II) or (III),in certain embodiments W and Z are joined to form a 6-membered arylring.

Among the heteroaromatic nitrones described above by formula (I), (II),or (III), in certain embodiments W and Z are joined to form a 6-memberedheteroaryl ring. The heteroaryl ring can be any 5- to 8-memberedheteroaryl ring known to those of skill in the art. In certainembodiments, the heteroaryl ring is a pyridine, pyrimidine, furan,thiophene or pyrrole ring.

Referring to bicyclic aryl nitrones of formula (I), (II) or (III), incertain embodiments R¹ is substituted with a group other than phenyl,substituted phenyl or methyl. In other embodiments R¹ is substitutedwith a group other than phenyl, substituted phenyl or lower alkyl. Forinstance, R¹ can be substituted or unsubstituted heteroalkyl,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted aralkyl, or substituted orunsubstituted heteroaralkyl.

Also referring to bicyclic aryl nitrones of formula (I), (II) or (III),in certain embodiments R² can be substituted with a group other thanhydrogen. For instance, R² can be substituted or unsubstituted(C₁-C₆)alkyl, substituted or unsubstituted (C₁-C₆) cycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedaralkyl.

Referring again to bicyclic aryl nitrones of (I), (II) or in certainembodiments W and Z are joined to form a six-membered ring that is fusedto a second ring. The second ring can be, for instance, a five- orsix-membered ring and can contain heteroatom(s). The second ring can befused to any adjacent pair of atoms in the first ring.

Also referring to bicyclic aryl nitrones of formulas (I), (II) or (III),in certain embodiments W and Z are joined to form a seven-membered ringthat is fused to a second ring. The second ring can be, for instance, afive-membered ring and can contain heteroatom(s). The second ring can befused to any adjacent pair of atoms in the first ring. For example, thebicyclic aromatic ring can be azulene.

In certain embodiments of aryl and heteroaromatic nitrones of formula(I), (II) or (III), W and X of Cy is C—R⁶. While the R⁶ substituents atW and X can vary independently, in certain embodiments both R⁶s areidentical. In particular embodiments, R⁶ are identical when it is SO₂R⁷or SO₃H.

Among the nitrone compounds of formulas (I), (II) or (III) in certainembodiments R² is hydrogen, alkyl, heteroalkyl, aralkyl or aryl, with orwithout further substitution. In some embodiments, R² is hydrogen.

In some embodiments, one or more of the R⁵ groups are hydrogen.

In some embodiments, R⁶ is hydrogen, —SR⁷, —SO₂R⁷, —SO₂NR⁷R⁸, —SO₃R⁷CONR⁷R⁸, —NR⁷R⁶, —OH, and —CO₂R⁷. In certain embodiments, R⁶ ishydrogen, —SO₂R⁷, —SO₂NR⁷R⁸, —SO₃R⁷, —CONR⁷R⁸, and —CO₂R⁷.

In the heteroaromatic nitrone compounds of the invention, the atomdesignated by X can be substituted or unsubstituted, especially incompounds where X is a carbon or a heteroatom with a free valence. Incertain embodiments, X can be substituted with any group other thanhydrogen. For instance, X can be substituted with —SR⁷, —SO₂R⁷,—SO₂NR⁷R⁸, —SO₃R⁷, —CONR⁷R⁸, —NR⁷R⁸, —OH, —PO(OR⁷)NR⁸R⁹, —PO(OR⁷)₂ or—CO₂R⁷.

Referring to heteroaromatic nitrone compounds of formulas (I), (II) or(III), in some embodiments for Cy the six-membered heteroaryl ringcontains one nitrogen atom, and in other embodiments the heteroaryl ringcontains two nitrogen atoms. In further embodiments, the ring containsthree nitrogen atoms.

When the heteroaryl ring (Cy) of formulas (I), (II) or (III) containstwo nitrogen atoms, the two nitrogen atoms can be at any of W, X, Y andZ. For instance, the two nitrogen atoms can be at W and X, at W and Y,at W and Z, at X and Y, at X and Z, or at Y and Z.

Among the bicyclic aryl nitrone compounds described by formulas (I),(II) or (III), in certain embodiments W and Z are joined to form a6-membered aryl or heteroaryl ring fused to a 5- or 6-memberedcycloalkyl, cycloheteroalkyl, aryl or heteroaryl ring.

Also among the bicyclic aryl nitrone compounds of the formulas above, insome embodiments R¹ is alkyl, cycloalkyl, aryl or aralkyl. In certainembodiments, R¹ is alkyl, including lower alkyl. In certain embodiments,the lower alkyl has branching at the 1-position carbon, for example,cyclopropyl, isopropyl, sec-butyl, tert-butyl, cyclobutyl,1-methylcycloprop-1-yl, sec-pentyl, tert-pentyl, cyclopentyl,1-methylcyclobut-1-yl and the like. In certain embodiments, R¹ istert-butyl.

In some embodiments, R² is hydrogen, alkyl, heteroalkyl, aralkyl oraryl, with or without further substitution.

In some embodiments, one or more R⁵ groups are hydrogen.

In some embodiments, R⁶ is hydrogen, —SR⁷, —SO₂R⁷, —SO₂NR⁷R⁸, —SO₃R⁷,—CONR⁷R⁸, —NR⁷R⁸, —OH, and —CO₂R⁷. In certain embodiments, R⁶ ishydrogen, —SO₂R⁷, —SO₂NR⁷R⁸, —SO₃R⁷, —CONR⁷R⁸, and —CO₂R⁷.

In the bicyclic aryl nitrone compounds of the invention, the atomdesignated by X can be substituted or unsubstituted, especially incompounds where X is a carbon or a heteroatom with a free valence. Incertain embodiments, X can be substituted with any group other thanhydrogen. For instance, X can be substituted with hydrogen, —SR⁷,—SO₂NR⁷R⁸, —SO₃R⁷, —CONR⁷R⁸, —NR⁷R⁸, —OH, —PO(OR⁷)NR⁸R⁹, —PO(OR⁷)₂, or—CO₂R⁷.

In certain embodiments, when the compound is formula (I), L is no atom,R¹ is tert-butyl, R² is hydrogen, and Cy is

wherein

R²¹ is R²² or R²²—S—, and

each R²² is independently selected from the group consisting ofhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted aryl, substituted or unsubstituted arylalkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedcycloheteroalkyl, substituted or unsubstituted heteroalkyl, substitutedor unsubstituted heteroaryl, and substituted or unsubstitutedheteroarylalkyl.

In certain embodiments, when the compound is formula (I), L is no atom,R¹ is tert-butyl, R² is hydrogen, and Cy is

wherein R²¹ is R²²—S—.

In certain embodiments, the compound is α-phenyl-tert-butyl nitrone(“PBN”), wherein the compound is formula (I), L is no atom, R¹ isPert-butyl, R² is hydrogen, and Cy is a benzene.

In certain embodiments, when the compound is formula (III), the compoundis symmetrical.

In certain embodiments, when the compound is formula (III), L is noatom, R¹ is tert-butyl, R² is hydrogen, and Cy is

wherein

each R²² is independently selected from the group consisting ofhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted aryl, substituted or unsubstituted arylalkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedcycloheteroalkyl, substituted or unsubstituted heteroalkyl, substitutedor unsubstituted heteroaryl, and substituted or unsubstitutedheteroarylalkyl. Compounds of this embodiment have the followingstructure:

and derivatives thereof, such as the salts, solvates, hydrates, andprodrug forms thereof, and spatial isomers thereof, as well aspharmaceutical preparations thereof.

In certain embodiments, when the thiol-modified nitrone compound is asymmetrical disulfide conjugate ofalpha-(4-sulfanylphenyl)-N-tert-butylnitrone, the compound is of formula(III), wherein L is no atom, R¹ is tert-butyl, R² is hydrogen, and Cy isbenzene, (also referred to herein as TK-115339), as shown below:

and derivatives thereof, such as the salts, solvates, hydrates, andprodrug forms thereof, and stereoisomers thereof, as well aspharmaceutical preparations thereof.

Thus in certain embodiments, the nitrone compounds are thiol-modifiednitrone compounds, such as the disulfide conjugates depicted in formula(III), as well as certain compounds of formula (I) wherein Cy comprisesa thiol group or thiol conjugate thereof, such as with compounds offormula (II).

By “thiol conjugate” is intended any compound or molecule capable ofconjugation to a thiol group. Examples of thiol conjugates include, butare not limited to, compounds or molecules that react with the thiol toform a bond selected from disulfide, thioether, thioacetal and thioester(see, e.g., “March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 6th Edition,” Michael B. Smith, Jerry March, 2007, JohnWiley & Sons Inc.). This includes thiol protecting groups (see, e.g.,“Greene's Protective Groups in Organic Synthesis, 4th Edition,” TheodoraW. Greene, Peter G. M. Wuts, 2006, John Wiley & Sons Inc; and “March'sAdvanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6thEdition,” Michael B. Smith, Jerry March, 2007, John Wiley & Sons Inc.).Of specific interest are thiol-modified nitrone compounds that aredisulfide conjugates of formula (I).

In certain embodiments, when the thiol-modified nitrone compound isformula (I), Cy is RS-Cy, and is a compound of formula (II), wherein R¹,R², L and Cy are as defined above, and R is hydrogen or a thiolconjugate.

In certain embodiments, when the thiol-modified nitrone compound isformula (II), Cy is

wherein R²² is as defined above.

In certain embodiments, when the thiol-modified nitrone compound isformula (II), L is no atom, R¹ is tert-butyl, R² is hydrogen, and Cy is

wherein R²² is as defined above. Compounds of this embodiment have thefollowing structure:

and derivatives thereof, such as the salts, solvates, hydrates, andprodrug forms thereof, and spatial isomers thereof, as well aspharmaceutical preparations thereof.

In certain embodiments, when the thiol-modified nitrone compound isformula (II), L is no atom, R¹ is tert-butyl, R² is hydrogen, and Cy is

wherein each R²² is hydrogen. Compounds of this embodiment have thefollowing structure:

and derivatives thereof, such as the salts, solvates, hydrates, andprodrug forms thereof, and spatial isomers thereof, as well aspharmaceutical preparations thereof.

In certain embodiments, when the thiol-modified nitrone compound isformula (II), R is R²².

In certain embodiments, when the thiol-modified nitrone compound isalpha-(4-sulfanylphenyl)-N-tert-butylnitrone, the compound is of formula(II), wherein L is no atom, R¹ is tart-butyl, R² is hydrogen, Cy isbenzene, and R is hydrogen, as shown below:

and derivatives thereof, such as the salts, solvates, hydrates, andprodrug forms thereof, and spatial isomers thereof, as well aspharmaceutical preparations thereof.

In certain embodiments of compounds of formula (II), the thiol-modifiednitrone compound is an non-symmetrical disulfide conjugate ofalpha-(4-sulfanylphenyl)-N-tert-butylnitrone, such as shown below.

In this example, the thiol-modified nitrone is coupled to a modifiedresveratrol, another potent anti-oxidant with anti-inflammatory,anti-cancer and neuroprotective effects (Rocha-González et al., CNSNeurosci Ther., 14: 234-47 (2008); Rhone M et al., Nutr Rev., 66: 465-72(2008); Udenigwe et al., Nutr Rev, 66: 445-54 (2008); Fan et al., Int JVitam Nutr Res.; 78: 3-8 (2008); Calabrese et al., Bellia et al.,Neurochem Res., 33: 2444-71 (2008); Singletary and Milner, CancerEpidemiol Biomarkers Prev., 17:1596-610 (2008); Jiang, Biochem BiophysRes Commun., 373, 341-4 (2008); Kundu and Surh, Cancer Lett., 269,243-61 (2008); Raval et al., Curr Med Chem.; 15, 1545-51 (2008)).

Another embodiment is a non-symmetrical disulfide conjugate ofalpha-(4-sulfanylphenyl)-N-tert-butylnitrone with ananti-thrombolytic/anti-inflammatory, such as salicylic acid as shownbelow.

Thus in certain embodiments, the nitrone compounds are thiol-modifiednitrone compounds, such as the disulfide conjugates, symmetrical andnon-symmetrical disulphides, depicted in formulas (II) and (III), aswell as certain compounds of formula (I) wherein Cy comprises a thiolgroup or thiol conjugate thereof. For example, nitrone compounds offormula (II) in which R is a thiol conjugate, and consisting of Cysubstituted with R¹, R² and/or L as defined above, the thiol conjugatemoiety is often an established pharmacophor or an agent added to modifyclearance or distribution of the molecule. In some embodiments, such asfor certain non-symmetrical disulphides of formula (II) or (III), thecompound coupled to the thiol group of the nitrone-containing moiety(e.g., alpha-(4-sulfanylphenyl)-N-tert-butylnitrone) can be biologicallyactive itself, or biologically inactive, provided only to modulate orotherwise improve or optimize one or more of the biopharmaceutical orpharmacokinetic (i.e., absorption, distribution, metabolism, and/orexcretion) characteristics of the compound.

As can be appreciated, derivatives of the above compounds include notonly the salts, solvates, hydrates, and prodrug forms thereof, andspatial isomers such as stereoisomers and geometric isomers thereof, aswell as pharmaceutical preparations thereof, suitable derivativesinclude detectably labeled versions of the subject nitrone compounds,which can be radiolabels, fluorophores, luminophores, and the like. Forinstance, thiol-conjugates include detectable labels attached to a thiolgroup of the subject thiol-modified nitrone compounds of the invention,and chemistries and labels for incorporation or attachment to thiolgroups are well known and commercially available.

The nitrone compounds described above and other nitrone derivatives arecommercially available or can be conventionally prepared by techniquesknown to one of skill in the art. The compounds of formula (II) or (III)can be synthesized by coupling thiolated nitrones or other suitablecoupling groups. For example, representative patents describing variousnitrone compounds and derivatives thereof, as well as thesynthesis/preparation thereof, include U.S. Pat. Nos. 3,849,934;4,596,874; 4,661,433; 4,758,669; 5,025,032; 5,036,097; 5,091,449;5,310,620; RE35,112; 5,475,032; 5,488,145; 5,498,778; 5,508,305;RE35,213; 5,723,502; 5,780,510; 5,849,771; 5,900,227; 5,942,507;5,972,977; 5,998,469; 6,015,831; 6,034,250; 6,040,444; 6,051,571;6,083,988; 6,083,989; 6,127,408; 6,140,356; 6,194,461; 6,197,825;6,197,826; 6,258,852; 6,291,702; 6,310,092; 6,342,523; 6,376,540;6,545,056; 6,569,902; 6,730,700; 6,762,322; 6,815,459; 6,835,754; and6,998,419; as well as published U.S. Application Pub. Nos. 2005/0059638,2005/0182060, 2005/0192281, and 2006/0100289; the disclosures of whichare herein incorporated by reference.

The doxorubicin toxicity-reducing adjuvants of interest can also includea nitrone compound in conjunction with a bisdioxopiperazine compound,and be employed in a pharmaceutical composition, kit or method of theinvention. For example, one such method involves administering aneffective amount of a doxorubicin active agent in conjunction with aneffective amount of a doxorubicin toxicity-reducing adjuvant, where thedoxorubicin toxicity-reducing adjuvant includes (i) a nitrone compoundor derivative thereof, and (ii) as an optional separate or admixedcomponent, a bisdioxopiperazine compound. Thus, pharmaceuticalcompositions and kits for practicing this aspect of the invention alsoare provided.

Examples of nitrone compounds of interest are those selected from:5,5-dimethyl-1-pyrroline-N-oxide; alpha-phenyl-N-tert-butyl nitrone;alpha-(2,4-disulfophenyl)-N-tert-butyl nitrone,alpha-(4-sulfanylphenyl)-N-tert-butylnitrone, and symmetrical disulfideconjugates of alpha-(4-sulfanylphenyl)-N-tert-butylnitrone. A nitronecompound of particular interest is alpha-phenyl-N-tert-butyl nitrone(“PBN”). Another nitrone compound of particular interest isalpha-(4-sulfanylphenyl)-N-tert-butylnitrone. Of special interest arecompounds comprising alpha-(4-sulfanylphenyl)-N-tert-butylnitrone andderivatives thereof, including conjugates thereof in which the sulfanylgroup is conjugated to a second compound, such as a symmetricaldisulfide conjugate of alpha-(4-sulfanylphenyl)-N-tert-butylnitrone(i.e., “TK-115339”), or a non-symmetrical disulfide conjugate ofalpha-(4-sulfanylphenyl)-N-tert-butylnitrone, such asalpha-(4-sulfanylphenyl)-N-tert-butylnitrone conjugated through adisulfide to a thiol-modified resveratrol or salicylic acid.

Examples of bisdioxopiperazine compounds of interest are those selectedfrom: 4-[1-(3,5-dioxopiperazin-1-yl) propan-2-yl]piperazine-2,6-dione(dexrazoxane); 4-[1-(3,5-dioxopiperazin-1-yl)ethan-2-yl]piperazine-2,6-dione; 4-[1-(3,5-dioxopiperazin-1-yl)1-methyl-butan-2-yl]piperazine-2,6-dione; 4-[1-(3,5-dioxopiperazin-1-yl)1-methyl-propan-2-yl]piperazine-2,6-dione; and4-[1-(3,5-dioxopiperazin-1-yl) butan-2-yl]piperazine-2,6-dione. Abisdioxopiperazine compound of interest is4-[1-(3,5-dioxopiperazin-1-yl) propan-2-yl]piperazine-2,6-dione, whichis also referred to as “Dexrazoxane.”

In some embodiments, the nitrone compound is alpha-phenyl-N-tert-butylnitrone (i.e., “PBN”), and the bisdioxopiperazine compound is4-[1-(3,5-dioxopiperazin-1-yl) propan-2-yl]piperazine-2,6-dione (i.e.,“Dexrazoxane”). In other embodiments, the nitrone compound isalpha-(4-sulfanylphenyl)-N-tert-butylnitrone or a non-symmetricaldisulfide conjugate thereof, or a symmetrical disulfide conjugatethereof (i.e., “TK-115339”), and the bisdioxopiperazine compound isDexrazoxane.

Dexrazoxane is a prodrug analogue of the metal chelator EDTA thatprotects against anthracycline-induced cardiac toxicity, and most likelyacts by removing iron from the iron-doxorubicin complex, thus preventingformation of damaging reactive oxygen species (Cvetkovic et al., Drugs65(7):1005 (2005)). The anti-tumor efficacy of anthracyclines such asdoxorubicin is unlikely to be altered by dexrazoxane use ((Hochster etal., Semin Oncol. 25(4 Suppl 10):37 (1998); and Marty et al., Ann Oncol.17(4):614 (2006)). Also, dexrazoxane appears to be useful for treatingaccidental extravasation injury from the use of the anthracyclineanticancer drugs doxorubicin, daunorubicin, epirubicin and idarubicin,which can be a serious complication of their use (Hasinoff, B B, FutureOncol., 2(1):15-20 (2006). Accordingly, it is believed that thecombination of a nitrone compound with a bisdioxopiperazine compound canfurther benefit a patient to reduce the unwanted side effects ofdoxorubicin active agents.

The bisdioxopiperazine compounds described above and otherbisdioxopiperazine derivatives are commercially available or can beconventionally prepared by techniques known to one of skill in the art.For example, representative patents describing various nitrone compoundsand derivatives thereof, as well as the synthesis/preparation thereof,include U.S. Pat. Nos. 3,941,790; 4,755,619; 4,764,614; 4,902,714;4,943,578; 4,963,551; 4,963,679; 5,149,710; 5,162,372; 5,242,901;5,278,187; 5,438,057; 5,492,913; 5,618,936; 5,688,797; 5,760,039; and6,693,100; the disclosures of which are herein incorporated byreference. In certain embodiments, the nitrone compound is not2,4-disulfonyl α-phenyl tertiary butyl nitrone, e.g., as disclosed inU.S. Pat. No. 5,508,305.

As indicated above, an effective amount of toxicity-reducing adjuvant isemployed in the subject methods. In certain embodiments, the amount oftoxicity-reducing adjuvant employed is not more than about the amount ofthe doxorubicin active agent employed. In certain embodiments, theamount of toxicity-reducing adjuvant employed is an amount that is lessthan equi-molar to the amount of doxorubicin active agent that isadministered. Typically, the amount of toxicity-reducing adjuvant thatis administered is less than about 75%, less than about 50%, less thenabout 25% and many embodiments less than about 15%, less than about 10%and even less than about 5% or 1% than the amount of doxorubicin activeagent. In other embodiments, the effective amount is the same as theamount of the active agent, and in certain embodiments the effectiveamount is an amount that is more than the amount of the doxorubicinactive agent. Effective amounts can readily be determined empiricallyusing the data provided in the Experimental section, below.

Standard dosing regiments can be employed in which the doxorubicintoxicity-reducing adjuvant is administered within the window oftherapeutic opportunity. For example, one regimen for administering adoxorubicin toxicity-reducing adjuvant is from about 6 to 12 hoursbefore to about 6 to 12 hours after the start of a particulardoxorubicin dosing regimen, such as from about 3 to 6 hours before toabout 3 to 6 hours after the start of a doxorubicin dosing regimen. Thusin one embodiment, a doxorubicin toxicity-reducing adjuvant is given toa patient in need thereof within the window of therapeutic opportunityfor that patient, i.e., within a few hours before or after the start ofa doxorubicin dosing regimen.

Optimal dosing strategies also can be employed in which dosing isindividualized based on a metabolite clearance parameter or bodyweight,estimated using the population pharmacokinetic models, empiricalcovariate distributions relevant for the target population, and a targetdefinition based on target fulfillment criteria and parsimony. Forinstance, the doxorubicin toxicity-reducing adjuvant (as separate orcombined components) can be administered as a loading dose to reach aneffective plasma concentration over a short period of time, andoptionally, followed by one or more subsequent (or continuous) doses tomaintain the desired plasma level for a given period of time. Aparticular example of a loading-maintenance dose approach is where adoxorubicin toxicity-reducing adjuvant is administered as a loading doseover about 1 to 2 hours, followed by a maintenance dose(s) for about 24to 72 hours. Thus, loading as well as maintenance or standard dosing canbe individualized based on pharmacokinetic parameters, such as themonitoring clearance of a particular metabolite, in conjunction withcut-off values at which dosing amounts and rates are incremented ordecremented. It will be appreciated that dosing regiment can includeintermittent recovery periods, such as recovery periods between thevarious treatments.

The scope of the present invention includes prodrugs of the doxorubicinactive agent and doxorubicin toxicity-reducing adjuvant. Such prodrugsare, in general, functional derivatives of the compounds that arereadily convertible in vivo into the required compounds. Thus, in themethods of the present invention, the term “administering” encompassesadministering the compound specifically disclosed or with a compoundwhich may not be specifically disclosed, but which converts to thespecified compound in vivo after administration to the subject in needthereof. Conventional procedures for the selection and preparation ofsuitable prodrug derivatives are described, e.g., in Wermuth, “DesigningProdrugs and Bioprecursors” in Wermuth, ed. The Practice of MedicinalChemistry, 2d Ed., pp. 561-586 (Academic Press 2003). Prodrugs includeesters that hydrolyze in vivo (e.g., in the human body) to produce acompound described herein suitable for the present invention. Suitableester groups include, without limitation, those derived frompharmaceutically acceptable, aliphatic carboxylic acids, particularlyalkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which eachalkyl or alkenyl moiety has no more than 6 carbon atoms. Illustrativeesters include formates, acetates, propionates, butyrates, acrylates,citrates, succinates, and ethylsuccinates.

Formulations

Also provided are pharmaceutical compositions containing the doxorubicinactive agent and/or doxorubicin toxicity-reducing adjuvant employed inthe subject methods. Accordingly, the doxorubicin active agent and/ordoxorubicin toxicity-reducing adjuvant can be formulated for oral orparenteral administration for use in the subject methods, e.g., in theform of a pharmaceutically acceptable salt, as described above. Incertain embodiments, e.g., where the compounds are administered asseparate formulations (such as in those embodiments where they areadministered sequentially), separate or distinct pharmaceuticalcompositions, each containing a different active agent, are provided. Inyet other embodiments, a single formulation that includes both of thedoxorubicin active agent and/or doxorubicin toxicity-reducing adjuvant(i.e., one composition that includes both active agents) is provided.

By way of illustration, the doxorubicin active agent and/or doxorubicintoxicity-reducing adjuvant can be admixed with conventionalpharmaceutically acceptable carriers and excipients (i.e., vehicles) andused in the form of aqueous solutions, tablets, capsules, elixirs,suspensions, syrups, wafers, and the like. Such pharmaceuticalcompositions contain, in certain embodiments, from about 0.1 to about90% by weight of the active compound, and more generally from about 1 toabout 30% by weight of the active compound. The pharmaceuticalcompositions may contain common carriers and excipients, such as cornstarch or gelatin, lactose, dextrose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, andalginic acid. Disintegrators commonly used in the formulations of thisinvention include croscarmellose, microcrystalline cellulose, cornstarch, sodium starch glycolate and alginic acid.

A liquid composition will generally consist of a suspension or solutionof the compound or pharmaceutically acceptable salt in a suitable liquidcarrier(s), for example, ethanol, glycerine, sorbitol, non-aqueoussolvent such as polyethylene glycol, oils or water, with a suspendingagent, preservative, surfactant, wetting agent, flavoring or coloringagent. Alternatively, a liquid formulation can be prepared from areconstitutable powder.

For example, a powder containing active compound, suspending agent,sucrose and a sweetener can be reconstituted with water to form asuspension, and a syrup can be prepared from a powder containing activeingredient, sucrose and a sweetener.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidcompositions. Examples of such carriers include magnesium stearate,starch, lactose, sucrose, microcrystalline cellulose and binders, forexample, polyvinylpyrrolidone. The tablet can also be provided with acolor film coating, or color included as part of the carrier(s). Inaddition, active compound can be formulated in a controlled releasedosage form as a tablet comprising a hydrophilic or hydrophobic matrix.

A composition in the form of a capsule can be prepared using routineencapsulation procedures, for example, by incorporation of activecompound and excipients into a hard gelatin capsule. Alternatively, asemi-solid matrix of active compound and high molecular weightpolyethylene glycol can be prepared and filled into a hard gelatincapsule; or a solution of active compound in polyethylene glycol or asuspension in edible oil; for example, liquid paraffin or fractionatedcoconut oil can be prepared and filled into a soft gelatin capsule.

Tablet binders that can be included are acacia, methylcellulose, sodiumcarboxymethylcellulose, poly-vinylpyrrolidone (Povidone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose. Lubricants that canbe used include magnesium stearate or other metallic stearates, stearicacid, silicone fluid, talc, waxes, oils and colloidal silica.

Flavoring agents such as peppermint, oil of wintergreen, cherryflavoring or the like can also be used. Additionally, it may bedesirable to add a coloring agent to make the dosage form moreattractive in appearance or to help identify the product.

The compounds of the invention and their pharmaceutically acceptablesalts that are active when given parenterally can be formulated forintramuscular, intrathecal, or intravenous administration.

A typical composition for intramuscular or intrathecal administrationwill be of a suspension or solution of active ingredient in an oil, forexample, arachis oil or sesame oil. A typical composition forintravenous or intrathecal administration will be a sterile isotonicaqueous solution containing, for example, active ingredient and dextroseor sodium chloride, or a mixture of dextrose and sodium chloride. Otherexamples are lactated Ringer's injection, lactated Ringer's plusdextrose injection, Normosol-M and dextrose, Isolyte E, acylatedRinger's injection, and the like. Optionally, a co-solvent, for example,polyethylene glycol, a chelating agent, for example, ethylenediaminetetracetic acid, and an antioxidant, for example, sodium metabisulphitemay be included in the formulation. Alternatively, the solution can befreeze dried and then reconstituted with a suitable solvent just priorto administration.

The compounds of the invention and their pharmaceutically acceptablesalts which are active on rectal administration can be formulated assuppositories. A typical suppository formulation will generally consistof active ingredient with a binding and/or lubricating agent such as agelatin or cocoa butter or other low melting vegetable or synthetic waxor fat.

The compounds of this invention and their pharmaceutically acceptablesalts which are active on topical administration can be formulated astransdermal compositions or transdermal delivery devices (“patches”).Such compositions include, for example, a backing, active compoundreservoir, a control membrane, liner and contact adhesive. Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art (see, e.g.,U.S. Pat. No. 5,023,252, herein incorporated by reference in itsentirety). Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

In certain embodiments of interest, the doxorubicin active agent and thetoxicity-reducing adjuvant are administered as a single pharmaceuticalformulation, that, in addition to including an effective amount of theactive agent and toxicity-reducing adjuvant, includes other suitablecompounds and carriers, and also may be used in combination with otheractive agents. The present invention, therefore, also includespharmaceutical compositions comprising pharmaceutically acceptableexcipients. The pharmaceutically acceptable excipients include, forexample, any suitable vehicles, adjuvants, carriers or diluents, and arereadily available to the public. The pharmaceutical compositions of thepresent invention may further contain other active agents as are wellknown in the art.

One skilled in the art will appreciate that a variety of suitablemethods of administering a formulation of the present invention to asubject or host, e.g., patient, in need thereof, are available, and,although more than one route can be used to administer a particularformulation, a particular route can provide a more immediate and moreeffective reaction than another route. Pharmaceutically acceptableexcipients are also well-known to those who are skilled in the art, andare readily available. The choice of excipient will be determined inpart by the particular compound, as well as by the particular methodused to administer the composition. Accordingly, there are a widevariety of suitable formulations of the pharmaceutical composition ofthe present invention. The following methods and excipients are merelyexemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachetsor tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin, or sucroseand acacia, emulsions, gels, and the like containing, in addition to theactive ingredient, such excipients as are known in the art to beappropriate.

The subject formulations of the present invention can be made intoaerosol formulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They mayalso be formulated as pharmaceuticals for non-pressured preparationssuch as for use in a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Formulations suitable for topical administration may be presented ascreams, gels, pastes, or foams, containing, in addition to the activeingredient, such carriers as are known in the art to be appropriate.

Suppository formulations are also provided by mixing with a variety ofbases such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration may be presented as pessaries,tampons, creams, gels, pastes, foams.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the nature of the deliveryvehicle, and the like. Suitable dosages for a given compound are readilydeterminable by those of skill in the art by a variety of means.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to elicit a prophylacticor therapeutic response in the animal over a reasonable time frame. Oneskilled in the art will recognize that dosage will depend on a varietyof factors including the strength of the particular compound employed,the condition of the animal, and the body weight of the animal, as wellas the severity of the illness and the stage of the disease. The size ofthe dose will also be determined by the existence, nature, and extent ofany adverse side-effects that might accompany the administration of aparticular compound. Suitable doses and dosage regimens can bedetermined by comparisons to anticancer or immunosuppressive agents thatare known to elicit the desired growth inhibitory or immunosuppressiveresponse. In the treatment of some individuals with the compounds of thepresent invention, it may be desirable to use a high dose regimen inconjunction with a rescue agent for non-malignant cells. In suchtreatment, any agent capable of rescue of non-malignant cells can beemployed, such as citrovorum factor, folate derivatives, or leucovorin.Such rescue agents are well known to those of ordinary skill in the art.Rescue agents include those which do not interfere with the ability ofthe present inventive compounds to modulate cellular function.

Optionally, the pharmaceutical composition may contain otherpharmaceutically acceptable components, such a buffers, surfactants,antioxidants, viscosity modifying agents, preservatives and the like.Each of these components is well-known in the art (see, e.g., U.S. Pat.No. 5,985,310, the disclosure of which is herein incorporated byreference).

In another embodiment, the aqueous cyclodextrin solution furthercomprises dextrose, e.g., about 5% dextrose. Other components suitablefor use in the formulations of the present invention can be found inRemington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985).

Utility

The subject methods find use in therapeutic applications in whichdoxorubicin administration is indicated. A representative therapeuticapplication is the treatment of cellular proliferative diseaseconditions, e.g., cancers and related conditions characterized byabnormal cellular proliferation concomitant. Such disease conditionsinclude cancer/neoplastic diseases and other diseases characterized bythe presence of unwanted cellular proliferation, e.g., hyperplasias, andthe like. In these capacities, use of the present compositions willresult in reducing unwanted toxicity while retaining the desireddoxorubicin activity.

By treatment is meant that at least an amelioration of the symptomsassociated with the condition afflicting the host is achieved, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thecondition being treated. As such, treatment also includes situationswhere the pathological condition, or at least symptoms associatedtherewith, are completely inhibited, e.g., prevented from happening, orstopped, e.g. terminated, such that the host no longer suffers from theside-effects of the doxorubicin treatment or at least the symptoms thatcharacterize the side-effects.

A specific application of interest is the use of the nitrone compoundsof the invention to ameliorate doxorubicin-induced cardiotoxicity. Whenthe cumulative dose of doxorubicin reaches 450-550 mg/m², the risk ofdeveloping cardiac side effects, including congestive heart failure,dilated cardiomyopathy, and death, dramatically increase. Doxorubicincardiotoxicity can be characterized by a dose-dependent decline inmitochondrial oxidative phosphorylation. Reactive oxygen species,generated by the interaction of doxorubicin with iron, can then damagethe myocytes (heart cells), causing myofibrillar loss and cytoplasmicvacuolization. Additionally, some patients may develop “Hand-FootSyndrome” characterized by skin eruptions on the palms of the hand orsoles of the feet, characterized by swelling, pain and erythema.

Doxorubicin cardiotoxicity can also be characterized by certain subtypesof troponin (cardiac troponin I and T), which are very sensitive andspecific indicators of damage to the heart muscle (myocardium). Cardiacdamage results in elevated cardiac troponin levels in the blood. Thus,levels of cardiac troponin I and/or T can be easily measured in theblood or plasma to test for damaged heart muscle, including cardiacdamage resulting from myocardial infarction.

Accordingly, in certain embodiments, a method is provided for thetreatment of a host in need thereof an effective amount of a doxorubicinactive agent in conjunction with an amount of an doxorubicintoxicity-reducing adjuvant effective to reduce doxorubicin-inducedcardiotoxicity in the host, wherein the doxorubicin toxicity-reducingadjuvant is a nitrone compound of formulas (I), (II) or (III). In arelated embodiment, the doxorubicin-induced cardiotoxicity ischaracterized by one or more features selected from decline inmitochondrial oxidative phosphorylation and an increase in cardiactroponin levels. Of interest is the use of thiol-modified nitronecompounds of formulas (I), (II) and (III), and particularlythiol-modified nitrone compounds of formulas (II) and (III), and moreparticularly compound TK115339, as the doxorubicin toxicity-reducingadjuvant to reduce doxorubicin-induced cardiotoxicity in the subject.

Reduction of doxorubicin-induced cardiotoxicity is characterized by theprevention, mitigation, or reduction of the likelihood of onset ofcardiotoxicity resulting from treatment of a host with a doxorubicinactive agent. This includes treatment of a host in need thereof with aneffective amount of a doxorubicin active agent in conjunction with anamount of a doxorubicin toxicity-reducing adjuvant effective to reducedoxorubicin-induced cardiotoxicity in the host, where the doxorubicintoxicity-reducing adjuvant improves the likelihood of successfullypreventing or eliminating one or more features of cardiotoxicity when ithas occurred including: (i) prevention, that is, causing the clinicalsymptoms not to develop, e.g., preventing a decline in mitochondrialoxidative phosphorylation and/or damage to heart muscle, and/orpreventing progression of one or more of these features to a harmfulstate; (ii) inhibition, that is, arresting the development or furtherdevelopment of clinical symptoms, e.g., mitigating or completelyinhibiting an active (ongoing) feature of cardiotoxicity so that thefeature is decreased to the degree that it is no longer seriouslyharmful, which decrease can include complete elimination ofcardiotoxicity from the host; and/or (iii) relief, that is, causing theregression of clinical symptoms, e.g., causing a relief from a declinein mitochondrial oxidative phosphorylation and/or damage to heartmuscle, and/or other symptoms caused by treatment of the host with adoxorubicin active agent.

The thiol-modified nitrone compounds of formulas (I), (II) and (III),and particularly thiol-modified nitrone compounds of formulas (II) and(III), and more particularly compound TK115339, may also find use in thetreatment of other disorders amenable to nitrone compound-basedtherapies, such as described in U.S. Pat. Nos. 5,025,032; 5,036,097;5,622,994; 5,780,510; 6,083,988; 6,107,315; 6,1978,25; 6,291,702; and6,815,425.

A variety of subjects are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the subjects will be humans.

In certain embodiments, the subjects will be subjects that have beendiagnosed for and are therefore in need of administration of the activeagent. In certain embodiments, the methods may include diagnosing thesubject for the presence of the disease condition to be treated byadministration of the active agent.

The subject methods find use in, among other applications, the treatmentof cellular proliferative disease conditions, including neoplasticdisease conditions, i.e., cancers. In such applications, an effectiveamount of a doxorubicin active agent and a doxorubicin toxicity-reducingadjuvant are administered to the subject in need thereof. Treatment isused broadly as defined above, i.e., to include at least an ameliorationin one or more of the symptoms of the disease, as well as a completecessation thereof, as well as a reversal and/or complete removal of thedisease condition, i.e., a cure.

There are many disorders associated with a dysregulation of cellularproliferation, i.e., cellular hyperproliferative disorders.

Such conditions include those where there is proliferation and/ormigration of smooth muscle cells, and/or inflammatory cells into theintimal layer of a vessel, resulting in restricted blood flow throughthat vessel, i.e. neointimal occlusive lesions. Occlusive vascularconditions of interest include atherosclerosis, graft coronary vasculardisease after transplantation, vein graft stenosis, peri-anastomaticprosthetic graft stenosis, restenosis after angioplasty or stentplacement, and the like.

Other conditions of interest include diseases where there ishyperproliferation and tissue remodelling or repair of reproductivetissue, e.g. uterine, testicular and ovarian carcinomas, endometriosis,squamous and glandular epithelial carcinomas of the cervix, etc.

Tumors of interest for treatment include carcinomas, e.g. colon,duodenal, prostate, breast, melanoma, ductal, hepatic, pancreatic,renal, endometrial, stomach, dysplastic oral mucosa, polyposis, invasiveoral cancer, non-small cell lung carcinoma, transitional and squamouscell urinary carcinoma, etc.; neurological malignancies, e.g.neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhoodacute leukaemia, acute myelogenous leukemias, non-Hodgkin's lymphomas,chronic lymphocytic leukaemia, malignant cutaneous T-cells, mycosisfungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis,T-cell rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoidlupus erythematosus, lichen planus, etc.; and the like.

Some cancers of particular interest include breast cancers, which areprimarily adenocarcinoma subtypes. Ductal carcinoma in situ (DCIS) isthe most common type of noninvasive breast cancer. In DCIS, themalignant cells have not metastasized through the walls of the ductsinto the fatty tissue of the breast. Infiltrating (or invasive) ductalcarcinoma (IDC) has metastasized through the wall of the duct andinvaded the fatty tissue of the breast. Infiltrating (or invasive)lobular carcinoma (ILC) is similar to IDC, in that it has the potentialmetastasize elsewhere in the body. About 10% to 15% of invasive breastcancers are invasive lobular carcinomas.

Also of interest is non-small cell lung carcinoma. Non-small cell lungcancer (NSCLC) is made up of three general subtypes of lung cancer.Epidermoid carcinoma (also called squamous cell carcinoma) usuallystarts in one of the larger bronchial tubes and grows relatively slowly.The size of these tumors can range from very small to quite large.Adenocarcinoma starts growing near the outside surface of the lung andmay vary in both size and growth rate. Some slowly growingadenocarcinomas are described as alveolar cell cancer. Large cellcarcinoma starts near the surface of the lung, grows rapidly, and thegrowth is usually fairly large when diagnosed. Other less common formsof lung cancer are carcinoid, cylindroma, mucoepidermoid, and malignantmesothelioma.

Another disease of interest is melanoma which is a malignant tumor ofmelanocytes. Although most melanomas arise in the skin, they also mayarise from mucosal surfaces or at other sites to which neural crestcells migrate. Melanoma occurs predominantly in adults, and more thanhalf of the cases arise in apparently normal areas of the skin.Prognosis is affected by clinical and histological factors and byanatomic location of the lesion. Thickness and/or level of invasion ofthe melanoma, mitotic index, tumor infiltrating lymphocytes, andulceration or bleeding at the primary site affect the prognosis.Clinical staging is based on whether the tumor has spread to regionallymph nodes or distant sites. For disease clinically confined to theprimary site, the higher the chance of lymph node metastases and theworse the prognosis the greater the thickness and depth of localinvasion of the melanoma. Melanoma can spread by local extension(through lymphatics) and/or by hematogenous routes to distant sites. Anyorgan may be involved by metastases, but lungs and liver are commonsites.

Other hyperproliferative diseases of interest relate to epidermalhyperproliferation, tissue remodelling and repair. For example, thechronic skin inflammation of psoriasis is associated with hyperplasticepidermal keratinocytes as well as infiltrating mononuclear cells,including CD4+ memory T cells, neutrophils and macrophages.

The methods of the present invention can provide a highly general methodof treating many, if not most, malignancies, including tumors derivedfrom cells selected from skin, connective tissue, adipose, breast, lung,stomach, pancreas, ovary, cervix, uterus, kidney, bladder, colon,prostate, central nervous system (CNS), retina and blood, and the like.Representative cancers of interest include, but are not limited to:head/neck and lung tissue (e.g., head and neck squamous cell carcinoma,non-small cell lung carcinoma, and small cell lung carcinoma),gastrointestinal tract and pancreas (e.g., gastric carcinoma, colorectaladenoma, colorectal carcinoma, pancreatic carcinoma), hepatic tissue(e.g., hepatocellular carcinoma), kidney and urinary tract (e.g.,dysplastic urothelium, bladder carcinoma, renal carcinoma, Wilms tumor),breast (e.g., breast carcinoma), neural tissue (e.g., retinoblastoma,oligodendroglioma, neuroblastoma, malignant meningioma, skin (e.g.,normal epidermis, squamous cell carcinoma, basal cell carcinoma,melanoma, etc.), hematological tissues (e.g., lymphoma, chronic myeloidleukemia (CML), acute promyelocytic leukemia (APL), acute lymphoblasticleukemia (ALL), acute myeloid leukemia (AML), etc.), and the like.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to affect a prophylacticor therapeutic response in the animal over a reasonable time frame. Oneskilled in the art will recognize that dosage will depend on a varietyof factors including the strength of the particular compound employed,the dose of doxorubicin, the dosing regimen used for doxorubicin, thecondition of the animal, and the body weight of the animal, as well asthe severity of the illness and the stage of the disease.

The size of the dose will also be determined by the existence, nature,and extent of any adverse side-effects that might accompany theadministration of a particular compound.

Particular applications in which the subject methods and compositionsfind use include those described in U.S. Pat. Nos. 6,251,355; 6,224,883;6,130,245; 6,126,966; 6,077,545; 6,074,626; 6,046,044; 6,030,783;6,001,817; 5,922,689; 4,322,391; and 4,310,515; the disclosures of whichare herein incorporated by reference.

Additional applications in which the subject combination of doxorubicinactive agent and doxorubicin toxicity-reducing adjuvant find use includethose described further in U.S. Pat. No. 6,541,506 (such as coating ofmedical instruments or implants, agricultural applications, etc.) thedisclosure of which is herein incorporated by reference.

Kits & Systems

Also provided are kits and systems that find use in practicing thesubject methods, as described above. For example, kits and systems forpracticing the subject methods may include one or more pharmaceuticalformulations, which include one or both of the doxorubicin active agentand doxorubicin toxicity-reducing adjuvant. As such, in certainembodiments the kits may include a single pharmaceutical composition,present as one or more unit dosages, where the composition includes boththe doxorubicin active agent and doxorubicin toxicity-reducing adjuvant.In yet other embodiments, the kits may include two or more separatepharmaceutical compositions, each containing either a doxorubicin activeagent or a doxorubicin toxicity-reducing adjuvant.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., a diskette, a CD, etc., on which the information has beenrecorded. Yet another means that may be present is a website addresswhich may be used via the internet to access the information at a remotesite. Any convenient means may be present in the kits.

The term “system” as employed herein refers to a collection of adoxorubicin active agent and a doxorubicin toxicity-reducing adjuvant,present in a single or disparate composition, that are brought togetherfor the purpose of practicing the subject methods. For example,separately obtained doxorubicin active agent and doxorubicintoxicity-reducing adjuvant dosage forms brought together andco-administered to a subject, or administered sequentially, oradministered as part of another treatment regimen, according to thepresent invention, are a system according to the present invention.

The following examples further illustrate the present invention andshould not be construed as in any way limiting its scope.

EXPERIMENTAL RESULTS

I. Mouse Study

The efficacy of cancer chemotherapy can be improved if agents areavailable to minimize the adverse events associated with treatment withcytotoxics. The aim of this study was to find a dose of a nitronecompound that protects against doxorubicin-induced toxicity in a mouse.

To this end, a representative nitrone test article (TK-115339) wasexamined for its ability to mitigate one aspect of doxorubicin-inducedtoxicity, cardiac damage, in CD-1 mice as determined by concentration ofcardiac troponin I (cTnI) in animal plasma (Li et al., Circulation,113:535-43 (2006) and Hou et al., J Lab Clin Med., 146: 299-303 (2005).Doxorubicin was purchased from Xinchem Corporation (China). TK-115339was synthesized at the University of California, Santa Cruz.

Eight to ten-week-old male CD-1 male mice (Charles River, Hollister,Calif.) weighing 20-25 g were housed in a climate-controlledenvironment, the light was controlled (light:dark, 12:12), and theanimals given food and water ad libitum. The animals (10animals/treatment group) were dosed ip (200 μl volume) on day 1 withvehicle (pH 7.4 buffered saline), 25 mg/kg doxorubicin or 25 mg/kgdoxorubicin plus 1.0 or 3.0 mg/kg TK-115339 3 hours before, and after,the doxorubicin treatment. A 1 mL syringe and 27G1/2 needle was used forthe injection, and the total injection volume of either doxorubicin orTK-115339 was 200 μL.

On days 2, 3 and 4, the mice received a single ip injection of either 1or 3 mg/kg of TK-115339. On day 5 (96-hours post initial dosing withdoxorubicin), plasma samples were obtained from each animal byretro-orbital puncture using a 75 mm untreated plastic clad Hematocrittube. The mice were then euthanized by carbon dioxide suffocation. Theplasma samples were analyzed for cTnI using a commercial ELISA testaccording to the manufacture's recommendations (Life Diagnostics, Inc.,Cat. No. 2010-1-HSP).

Data was analyzed using SPSS software (SPSS Inc., Chicago, Ill.). TheMann-Whitney U test was used for comparison of population variancesfollowed by an independent samples T test.

A representative set of data is depicted in FIG. 1, which demonstratesthat TK-115339 protects mice from doxorubicin-induced cardiac damageestimated by measurement of plasma cardiac troponin. Representative ofthe data shown in FIG. 1, mice (10 animals/treatment group) were dosedip on Day 1 with vehicle (pH 7.4 buffered saline), 25 mg/kg doxorubicinip or 25 mg/kg doxorubicin ip plus 1.0 or 3.0 mg/kg TK-115339 3 hoursbefore, and after, the doxorubicin treatment. On days 2, 3 and 4, themice received a single ip injection of either 1 or 3 mg/kg of TK-115339.On Day 5 (96-hours post initial dosing with doxorubicin), plasma sampleswere obtained from each animal by retro-orbital eye bleeds and the micesacrificed. The plasma samples were analyzed for cTnI using a commercialELISA test for troponin I. Boxplots of troponin findings from the studyare provided in FIG. 1.

II. Mouse Dose Response Study

The aim of this study was to establish the dose response for theprotection afforded by TK-115339 against doxorubicin-associatedcardiotoxicity.

Eight to ten-week-old male CD-1 male mice (Charles River, Hollister,Calif.) weighing 30-35 g were housed in a climate-controlledenvironment, the light was controlled (light:dark, 12:12), and theanimals were given food and water ad libitum. The animals (12animals/treatment group) were dosed ip (200 μl volume) on day 1 with 25mg/kg doxorubicin or 25 mg/kg doxorubicin plus 0.0001, 0.001, 0.01, 0.1or 1.0 mg/kg TK-115339 3 hours before, and after, the doxorubicintreatment. A 1 mL syringe and 27G1/2 needle was used for the injection,and the total injection volume of either doxorubicin or TK-115339 was200 μL.

On days 2 and 3 the mice received a single ip injection of either saline(doxorubicin alone group) or TK-1153393 (dose ranging experimentalgroups). On day 5 (96-hours post initial dosing with doxorubicin),plasma samples were obtained from each animal by retro-orbital punctureusing a 75 mm untreated plastic clad Hematocrit tube. The mice were theneuthanized by carbon dioxide suffocation. The plasma samples wereanalyzed for cTnI using a commercial ELISA test according to themanufacture's recommendations (Life Diagnostics, Inc., Cat. No.2010-1-HSP).

Data was analyzed using SPSS software (SPSS Inc., Chicago, Ill.). TheMann-Whitney U test was used for comparison of population variancesfollowed by an independent samples T test.

A representative set of data are depicted in FIG. 2, which demonstratesthe protection afforded by TK-115339 from doxorubicin-induced cardiacdamage, estimated by measurement of plasma cardiac troponin, is doseproportional. Representative of the data shown in FIG. 2, mice (12animals/treatment group) were dosed ip on Day 1 with 25 mg/kgdoxorubicin or 25 mg/kg doxorubicin plus 0.0001, 0.001, 0.01, 0.1 or 1.0mg/kg TK-115339 3 hours before, and after, the doxorubicin treatment. OnDay 5 (96-hours post initial dosing with doxorubicin), plasma sampleswere obtained from each animal by retro-orbital eye bleeds and the micesacrificed. The plasma samples were analyzed for cTnI using a commercialELISA test. Boxplots of troponin findings from the study are provided inFIG. 2.

III. Cell Culture Study

The aim of this study was to examine whether or not a representativenitrone test article, TK-115339, interfered with the desired activity ofdoxorubicin in vitro.

The tumor cell line for the cell culture study was CCRF-CEM (HumanT-ALL, CCL-119) from ATCC, and cells were cultured in accordance withthe product information sheets replacing 75 cm² culture flasks with 56.7cm² Petri dishes and adding 1% penicillin-streptomycin solution and 1%GlutaMAX™ to the culture medium. The compound, TK-115339, was dissolvedin DMSO then diluted to obtain initial working solutions of 1, 10 and100 μM. In testing, 100-fold dilutions were made in culture media togive final assay concentrations of 0.01, 0.1 and 1.0 μM. Doxorubicin wasdissolved in sterile filtered PBS. Three 100× solutions were made togive final assay concentrations of 0.01, 0.1 and 1.0 μM.

Cells were thawed according to ATCC protocol and plated in 10 mL ofsterile filtered growth medium. Cells were sub-cultured 3 to 7 timesbefore starting the experiments. The cell suspension was diluted duringthe logarithmic growth phase at a ratio of 1 mL of cell suspension to 4mL of fresh growth media. Aliquots of 100 μL of this suspension wereplated in 96 well microtiter plates and grown under ATCC recommendedatmospheric conditions. After 24 hours, 100 μL of growth medium and 2 μLof test solution were added to each well for 72 hour incubation.Doxorubicin was evaluated at concentrations of 0.01, 0.1 and 1.0 μMalone or in combination with TK-115339 at concentrations of 0.01, 0.1and 1.0 μM. In addition, the same concentrations of TK-115339 weretested alone. For each condition, n=12.

At the end of incubation, cell viability was determined by opticalabsorbance of alamarBlue® at λ=570 and 600 nm in accordance using astandard protocol provided by the vendor, Biosource. Data was analyzedusing SPSS 14.0 software (SPSS Inc., Chicago, Ill.). Statisticalsignificance of the differences between experimental groups wascalculated by using one-way ANOVA followed by a Bonferroni post-hocanalysis.

Boxplots of typical results are provided in FIG. 3, which demonstratesthat TK-115339 does not interfere with the tumor cell anti-growthactivity of doxorubicin. Cells were prepared and tested as describedabove. Doxorubicin was evaluated at concentrations of 0.01, 0.1 and 1.0μM alone or in combination with TK-115339 at concentrations of 0.01, 0.1and 1.0 μM. TK-115339 was also tested alone at concentrations of 0.01,0.1 and 1.0 μM. For each condition, 12 replicates were analyzed. At theend of incubation, cell viability was determined by alamarBlue® usingoptical absorbance measurements at λ=570 and 600 nm. As shown in FIG. 3(Group 1=doxorubicin 0.01 μM; Group 2-4=doxorubicin 0.01 μM+TK-115339 at0.01, 0.1, and 1.0 μM; Groups 5=doxorubicin 0.1 μM; Group6-8=doxorubicin 0.1 μM+TK-115339 at 0.01, 0.1, and 1.0 μM; Group9=doxorubicin 1.0 μM; Groups 10-12=doxorubicin 1.0 μM+TK-115339 at 0.01,0.1, and 1.0 μM), TK-115339, a representative nitrone test article, doesnot interfere with the desired cytotoxicity of doxorubicin. Takentogether with the animal data, it is evident that nitrone compounds suchas TK-115339 are capable of reducing doxorubicin-induced toxicity whilenot significantly impacting the beneficial anti-neoplastic activity ofdoxorubicin.

It is evident from the above results and discussion that the subjectinvention provides for methods of reducing the unwanted toxicity ofdoxorubicin active agents while retaining their desired activity. Assuch, the subject invention finds use in a variety of differentapplications and represents a significant contribution to the art.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A method comprising: administering to a subjectan amount of a doxorubicin toxicity-reducing adjuvant effective toreduce toxicity of a doxorubicin active agent, wherein said doxorubicintoxicity-reducing adjuvant is a nitrone compound of formula (II):

or a pharmaceutically acceptable salt thereof, or a spatial isomerthereof: wherein: R is hydrogen or a disulfide conjugate; L is—[C(R³)₂]_(m)—X′—[C(R⁴)₂]_(n)—; m is an integer from 0 to 6; n is aninteger from 0 to 6; X′ is selected from the group consisting of noatom, NR₂, O, S, SO and SO₂; Cy is selected from the group consisting ofsubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted cycloheteroalkyl, bicycloalkenyl, bicycloheteroalkenyl,bicycloaryl, or bicycloheteroaryl ring; R¹ is selected from the groupconsisting of substituted or unsubstituted aliphatic, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted aralkyl, and substituted orunsubstituted heteroaralkyl; R² is selected from the group consisting ofhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, and substituted or unsubstituted aralkyl;R³ is selected from the group consisting of hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, and substituted or unsubstituted aralkyl; R⁴ is selectedfrom the group consisting of hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted aralkyl, and anytwo R³s may join together to form a cycloalkyl, cycloheteroalkyl ring;and one of R³s and one of R⁴s on carbon atoms adjacent to X′ together toform a heterocyclic ring of 5-7 atoms.
 2. The method according to claim1, wherein R is a disulfide conjugate, and said compound is of formula(Ill):

wherein R¹, R², L and Cy are each individually the same or different,and wherein said compound is symmetrical or non-symmetrical.
 3. Themethod according to claim 1, wherein L is no atom, R¹ is tert-butyl, R²is hydrogen, and Cy is

wherein R²¹ is R²²—S—, and each R²² is independently selected from thegroup consisting of hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkyl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted cycloheteroalkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heteroarylalkyl.
 4. The method according to claim 3,wherein each R²² is hydrogen.
 5. The method according to claim 2,wherein L is no atom, R¹ is tert-butyl, R² is hydrogen, and Cy is

wherein each R²² is independently selected from the group consisting ofhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkyl, substituted or unsubstituted arylalkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedcycloheteroalkyl, substituted or unsubstituted heteroalkyl, substitutedor unsubstituted heteroaryl, and substituted or unsubstitutedheteroarylalkyl.
 6. The method according to claim 5, wherein each R²² isindependently selected from the group consisting of hydrogen andhydroxyl.
 7. The method according to claim 1, wherein R is a disulfideconjugate that can be biologically active or biologically inactive, andis capable of modulating one or more of the biopharmaceutical and/orpharmacokinetic characteristics of said compound.
 8. The methodaccording to claim 1, wherein said compound is selected from the groupconsisting of:

or the pharmaceutically acceptable salts, solvates, hydrates, andprodrug forms thereof, spatial isomers thereof.
 9. The method accordingto claim 1, wherein said doxorubicin toxicity-reducing adjuvant isadministered to the subject at the same time as a doxorubicin activeagent.
 10. The method according to claim 1, wherein said doxorubicintoxicity-reducing adjuvant is administered to the subject sequentiallywith a doxorubicin active agent.
 11. The method according to claim 10,wherein said doxorubicin toxicity-reducing adjuvant is administered tothe subject prior to a doxorubicin active agent.
 12. The methodaccording to claim 10, wherein said doxorubicin toxicity-reducingadjuvant is administered to the subject after a doxorubicin activeagent.
 13. The method according to claim 1, wherein said doxorubicinactive agent is doxorubicin.
 14. The method according to claim 1,wherein said toxicity of said doxorubicin active agent iscardiotoxicity.